lysC定点突变及lysC、asdA串联表达对谷氨酸棒杆菌L-苏氨酸积累的影响

Effects of Site-directed Mutation of Gene lysC and Co-expression of lysC-asdA Cluster on L-threonine Accumulation in Corynebacterium glutamate

lysC、asdA基因分别编码的天冬氨酸激酶(Aspartate kinase,AK)和天冬氨酸半醛脱氢酶(Aspartate semi-aldehyde dehydrogenase,ASD)是L-苏氨酸合成途径中两个关键限速酶基因,其中AK受到代谢产物赖氨酸与苏氨酸的协同抑制。以选育获得的一株谷氨酸棒状杆菌T11(Corynebacterium glutamicum T11)为出发菌株,通过构建lysC-asdA串联表达盒,并对其关键限速酶基因lysC进行定点突变,突变位点为Ala279Thr,获得抗反馈抑制突变型编码基因lysCr-asdA,将其插入含强启动子tac的穿梭表达载体pZ8-1中成功构建串联表达质粒pZ8-1-lysCr-asdA转化出发菌株,筛选获得工程菌株T11/pZ8-1-lysCr-asdA。摇瓶发酵其L-苏氨酸产量达到7.18 g/L,较出发菌株提高27.8%。进一步的30 L发酵罐补料分批发酵结果显示,发酵60 h L-苏氨酸产量达65.5 g/L,糖酸转化率达到39.5%,较出发菌株分别提高29.5%和33.9%,为后续的进一步构建高产L-苏氨酸的谷氨酸棒杆菌工程菌株提供强有力的基础。

Aspartate kinase(AK)and aspartate semi-aldehyde dehydrogenase(ASD)encoded by gene lysC and gene asdA are two key rate-limiting enzymes playing vital roles in L-threonine synthesis pathway,and AK was co-inhibited by lysine and threonine.Selecting Corynebacterium glutamicum T11 as the original strain,the lysC-asdA gene co-expression cassette were constructed,the key rate-limiting enzyme gene lysC was mutated by site-directed mutation at Ala279Th,and the anti-feedback inhibition mutant gene,named lysC^r-asdA,was obtained,and was inserted into the shuttle expression vector pZ8-1 containing the strong promoter tac,thus the tandem expression plasmid pZ8-1-lysC^r-asdA was constructed successfully and transformed into the starting strain,then,the engineering strain T11/pZ8-1-lysC^r-asdA was obtained.The production of L-threonine by shaking flask was 7.18 g/L,which was 27.8% higher than that by the original strain.The results of further batch fermentation in 30 L fermenter showed that the yield of L-threonine reached 65.5 g/L for 60 h and the conversion rate of sugar and acid reached 39.5 g/L,increasing by 29.5% and 33.9% higher than by the original strain.It provides a strong basis for further constructing Corynebacterium glutamicum engineering strain highly yielding threonine.